NIS, the sodium iodide symporter, mediates the uptake and concentration of iodide in the thyroid gland, providing the basis for diagnostic thyroid radioimaging and radioiodine therapy, which are used routinely in clinical and veterinary practice. When the NIS gene is introduced into non-thyroid cells, they can, like thyroid cells, be detected using iodide/pertechnetate radioimaging and/or destroyed using iodide radiotherapy. Imanis Life Sciences is developing NIS reporter gene technology for accurate, sensitive, high resolution monitoring of gene regulation and cell fate in living subjects. Unlike luciferase and GFP reporter genes, which rely on optical imaging, NIS can be used for quantitative expression monitoring in large animals and humans as well as in deep-seated mouse tissues because, unlike photons, gamma rays are minimally attenuated by the tissues through which they pass. Additional advantages of NIS are the ready availability of clinically approved NIS-specific radioisotopes, lack of immunogenicity (NIS is a self-protein) and harmlessness to targeted tissues. Background signals from circulating radiotracer and from tissues naturally expressing NIS, such as thyroid, stomach and salivary glands, can significantly limit the sensitivity, resolution and utility of the NIS reporter gene technology. Importantly, NIS-mediated uptake of NIS specific radiotracer anions can be potently inhibited by perchlorate, which can be safely administered to human subjects for this purpose. The goal of the current application is therefore to isolate perchlorate resistant NIS mutants that efficiently transport iodide or pertechnatate ion even in the presence of perchlorate. Background signal in SPECT and PET imaging studies will then be reduced by coadministration of nonradioactive perchlorate anions with a NIS radiotracer. If the perchlorate-resistant NIS reporter protein is ion selective, and resistant to perchlorate inhibition, tracer uptake will occur in genetically modified cells/tissue expressing th mutated NIS protein, but be blocked in organs naturally expressing NIS. For the phase I application we have the following specific aims. SPECIFIC AIM 1: Screen cells expressing a library of NIS variants for a variant whose ability to transport iodide or pertechnetate is relativly resistant to perchlorate inhibition SPECIFIC AIM 2: To construct and validate substrate and co-transported ions binding sites in human NIS and rationally design mutations that will allow radioiodide or pertechnetate transport in the presence of perchlorate. The milestone for aim 1 or 2 that will trigger submission of phase II SBIR grant application will be the isolation of a NIS variant that transports at least 40% more iodide than wild-type (WT) NIS in the presence of perchlorate. The aim is highly feasible as we have already a first generation NIS variant that selectively uptakes NIS substrates.